CN114219715B - Quality enhancement method and system for structured illumination light super-resolution microscope image - Google Patents
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Abstract
The invention discloses a method and a system for enhancing the quality of a structured illumination light super-resolution microscope image, wherein the method comprises the following steps: step 1, carrying out frequency spectrum separation on an original SIM image to obtain different frequency spectrum components; step 2, carrying out frequency domain filtering on the frequency spectrum component, removing the defocusing information distributed in the low-frequency region of the frequency spectrum to remove the streak artifact and obtain the frequency spectrum component
Where d represents the direction of the structured illumination light, d is 1, 2, 3, m represents whether the spectral component is to be moved, m is 0 represents that the spectral component is not to be moved, and m ± 1 represents that the spectral component distribution is in accordance with ± pdIs moved, pdDenotes a structured light wave vector, k ═ k (k)x，ky)，kxAnd kyRespectively representing the spatial coordinates of the image frequency domain; step 3, mixing the above
According to mpdMoving to obtain the moved frequency spectrum component
Step 4, for the above
Performing spectrum combination; meanwhile, the optical propagation function is modulated to inhibit the defocused background, and finally the SIM super-resolution image is output. The method can not only effectively remove the artifact effect, but also inhibit the defocused background.
Description
Technical Field
The invention relates to the technical field of image quality enhancement, in particular to a method and a system for enhancing the quality of a structured illumination light super-resolution microscope image.
Background
The Structured light Illumination super-resolution microscope (SIM) imaging technology is an important imaging technology in the field of life science. In fluorescence microscopy, the illumination light excites the entire sample, but only focuses at a certain depth when the fluorescence signal is collected. If strong fluorophores outside the focal plane are excited, clustered out-of-focus information can be presented in the image, and the image signal-to-back ratio is reduced. Meanwhile, after the stronger defocus information and the sample signal are subjected to spectrum separation, spectrum shift and spectrum combination, 6 bright spots are easy to appear at the high-frequency position of the frequency domain of the reconstructed image, and appear as periodic streak artifacts or cellular artifacts on the spatial domain image. Therefore, the defocused information not only reduces the signal-to-back ratio of the SIM super-resolution image, but also may bring about artifacts and reduce the image quality.
The existing research for removing the defocus information mainly comprises the following steps: the method comprises the following steps of (I) using a high-pass filter to suppress out-of-focus information in an image reconstruction process; and (II) removing the defocusing information by using a half-edge filter.
Most of the defocusing information is distributed in the low-frequency area of the image, and is finally reflected as the bright spot at the high-frequency position of the image frequency spectrum through frequency spectrum separation, frequency spectrum shift and frequency spectrum combination. Zeiss SIM2(Dual Iterative SIM) uses a high-pass filter to suppress out-of-focus information during reconstruction.
The image is filtered by using a high-pass filter, and the value of the central area of the high-pass filter is 0, so that the frequency spectrum information of the zero-frequency area is all 0, and further the frequency spectrum information of the image is lost. In addition, improper setting of the high pass filter radius parameter may also create a partial signal loss problem.
DONGLI XU et al propose a method of using a half-edge filter in a Nonlinear SIM (NSIM) to suppress the out-of-focus background. The method assumes that the frequency spectrum of the defocus information is located in the low-frequency region of the image, and is only located in a half region of the image frequency spectrum after the image frequency spectrum is moved. Therefore, the partial frequency spectrum components containing the defocusing information can be removed through half-edge filtering, and then the filtered frequency spectrum components are symmetrically filled according to the characteristic that the frequency spectrum information has conjugate symmetry, so that the image frequency spectrum information without the defocusing information is obtained.
NSIM uses a half-edge filter to remove the defocus information implies the following assumptions:
(1) because of the diffusion function caused by defocus or scattering, defocus information exists only on one side of the spectrum after the defocus information of the spectral components is shifted;
(2) the left half signal spectrum and the right half signal spectrum of each spectral component are conjugate symmetric.
However, the defocus information located in the low-frequency region of the spectral components in the actual SIM imaging process is not only contained in one side of the spectral components after the spectral separation and shifting, and thus the defocus information cannot be completely removed by using the half-edge filter. In addition, the defocusing information after the spectrum movement is located in a high-frequency region of the spectrum, and is represented as a high-frequency bright point in the frequency domain, and is represented as an image with periodic artifacts in a space domain.
Disclosure of Invention
The invention aims to provide a quality enhancement method and a quality enhancement system for a structured illumination light super-resolution microscope image, which can effectively remove the artifact effect and can inhibit the defocused background.
In order to achieve the above object, the present invention provides a method for enhancing quality of a structured-illumination light super-resolution microscope image, comprising:
step 2, carrying out frequency domain filtering on the frequency spectrum component, removing the defocusing information distributed in the low-frequency region of the frequency spectrum to remove the streak artifact and obtain the frequency spectrum component
Where d denotes a direction of the structured illumination light, d 1, 2, 3, m denotes whether the spectral component is to be moved, m 0 denotes that the spectral component is not to be moved, and m ± 1 denotes that the spectral component distribution is in accordance with ± pdIs moved, pdDenotes a structured light wave vector, k ═ k (k)x，ky)，kxAnd kyRespectively representing the spatial coordinates of the image frequency domain;
step 3, mixing the above
According to mpdMoving to obtain the moved frequency spectrum component
Step 4, for the above
Performing spectrum combination; meanwhile, the optical propagation function is modulated to inhibit the defocused background, and finally the SIM super-resolution image is output.
Further, the step 2 filters the spectral components with a first filter NF described by equation (1) or equation (2) or equation (3):
in the formula (I), the compound is shown in the specification,
a. b and c are both adjusting parameters of the filter, and n represents the order of the filter.
Further, the step 4 is realized by formula (4):
wherein S represents the SIM super-resolution image output in the step 4, FilterdenominatorRepresenting a second Filter, FilternumertorDenotes a third filter, Om(k+mpd) Represents the optical propagation function O (k) in terms of mpdThe result after shifting, A (k) represents apodization function, w represents reconstruction adjusting parameter, ifft {. cndot. } represents fast FourierAnd (4) performing leaf inverse transformation.
Further, in the step 4, a FilterdenominatorIs set to the formula (5), FilternumertorIs set to formula (6):
wherein a, b and c each represent a filter adjustment parameter,
representing the square of the vector two norm.
Further, in the step 4, a FilterdenominatorIs set to the formula (7), FilternumertorIs set to formula (8):
in the formula, b represents the filter adjusting parameter, | ·| non-woven phosphor2Representing the two-norm of the vector.
Further, in the step 4, a FilterdenominatorIs set to the formula (9), FilternumertorIs set to formula (10):
in the formula, b represents the filter adjusting parameter, | ·| non-woven phosphor2Representing two norms of the vector, | · | | non-woven phosphor2Representing the two-norm of the vector.
The invention also provides a quality enhancement system for the structured illumination light super-resolution microscope image, which comprises:
a spectrum separation unit for obtaining different spectrum components by performing spectrum separation on the SIM original image;
a first filter for performing frequency domain filtering on the frequency spectrum component to remove the defocus information distributed in the low frequency region of the frequency spectrum to remove the streak artifact and obtain the frequency spectrum component
Where d represents the direction of the structured illumination light, d is 1, 2, 3, m represents whether the spectral component is to be moved, m is 0 represents that the spectral component is not to be moved, and m ± 1 represents that the spectral component distribution is in accordance with ± pdIs moved, pdDenotes a structured light wave vector, k ═ k (k)x，ky)，kxAnd kyRespectively representing image frequency domain space coordinates;
a spectrum moving unit for moving the spectrum
According to mpdMoving to obtain the moved frequency spectrum component
A reconstruction image unit for reconstructing the image
Carrying out frequency spectrum combination; meanwhile, the optical propagation function is modulated to inhibit the defocused background, and finally the SIM super-resolution image is output.
Further, the first filter NF is set to equation (1) or (2) or (3):
in the formula (I), the compound is shown in the specification,
a. b and c are both adjusting parameters of the filter, and n represents the order of the filter.
Further, the reconstructed image unit is set to equation (4):
wherein S represents the SIM super-resolution image output by the reconstruction image unit, FilterdenominatorRepresenting a second Filter, FilternumertorDenotes a third filter, k ═ k (k)x，ky) Wherein k isxAnd kyRespectively represent the image frequency domain space coordinates, Om(k+mpd) Represents the optical propagation function O (k) in terms of mpdAnd (b) the shifted propagation function, A (k) represents an apodization function, w represents a reconstruction adjusting parameter, and ifft {. cndot.) represents an inverse fast Fourier transform.
Further, the Filterdenominator、FilternumertorTo equations (5) and (6), respectively, or to equations (7) and (8), respectively, or to equations (9) and (10), respectively:
wherein a, b and c each represent a filter adjustment parameter,
represents the square of the vector two-norm, | ·| non-woven phosphor2Representing the two-norm of the vector.
Due to the adoption of the technical scheme, the invention has the following advantages: the method not only can effectively remove the artifact effect and inhibit the effect of the defocused background, but also has the advantages of low space occupancy rate and high processing speed, and can be suitable for application scenes with real-time processing requirements.
Drawings
Fig. 1 is a schematic flow chart of an image quality enhancement method for a structured-illumination light super-resolution microscope according to an embodiment of the present invention.
Fig. 2 is a schematic diagram of removing a streak artifact according to an embodiment of the present invention, where: a shows spectral components after spectral separation, b shows a filter corresponding to each spectral component, and c shows the result after filtering of the spectral components, in which out-of-focus information at high frequency positions in the frequency domain has been removed (black circles).
FIG. 3 is the present inventionIllustrative embodiments modulate a schematic diagram of an OTF, wherein: a shows the actual combined | OTF & ltmu & gt in SIM reconstruction2As a result, b shows the theory | OTF-2As a result, c shows the gray scale curve of the black straight line in a and b, in which the black arrows indicate regions where the two have significant difference.
Detailed Description
The invention is described in detail below with reference to the figures and examples.
As shown in fig. 1, a method for enhancing quality of a SIM image according to an embodiment of the present invention includes:
After the SIM original image is subjected to frequency spectrum separation, different frequency spectrum components can be obtained
d represents the direction of the structured illumination light, d is 1, 2, 3, m represents whether the spectral components are to be moved, m is 0, +1, -1, where: when m is 0, it means that the spectral component does not move, and m +1 means that the spectral component illuminates the light wave vector + p according to the structuredMoving, m-1 denotes spectral components illuminating light wave vector-p according to the structuredAnd (4) moving.
The moved spectrum components are represented, and the defocusing information originally located in the low-frequency area is moved to the high-frequency position of the spectrum of the reconstructed image, which shows that the reconstructed super-resolution image has streak artifacts. For spectral components that do not need to be moved
The defocusing information is still located in the low-frequency area of the reconstructed super-resolution image frequency spectrum and is reflected as the defocusing background of the reconstructed super-resolution image.
Step 2, filtering the frequency spectrum component, and filtering the frequency spectrum component
In one embodiment, step 2 filters the spectral components with a first filter NF described by equation (1):
the first filter NF may also be set to equation (2):
the first filter NF may also be set to equation (3):
in the formula (I), the compound is shown in the specification,
wherein k isxAnd kyRespectively representing the spatial coordinates of the frequency domain of the image, a, b and c are all the adjusting parameters of the filter, n represents the order of the filter>0, where n is generally 6, m represents whether the spectral component is to be moved, m is 0 represents that the spectral component is not to be moved, and m ± 1 represents that the spectral component distribution is in ± pdIs moved, pdRepresenting the structured illumination light wave vector.
Of course, the operation of step 2 may also be implemented by using the first filter NF in other implementations.
This embodiment is implemented by aligning spectral components
Filtering is carried out, and then the subsequent reconstruction step is carried out, so that the purpose of removing the streak artifact is achieved. As shown in fig. 2, a in the figure is an image spectrum component, b in the figure is a filter corresponding to each spectrum component, and c in the figure is an image frequency after filtering processing and complete reconstruction stepSpectrum, circles indicate that the corresponding defocus information has been filtered out. The defocus information is basically distributed in the low-frequency region of the spectrum, so that the defocus information is removed by frequency domain filtering before the image spectrum components are moved.
Step 3, performing spectrum movement on the spectrum components, for example:
according to mpdThe shifted spectral components are represented as
Step 4, in the reconstruction process, the invention also modulates an Optical Transfer Function (OTF), and the main purpose of the invention is to modulate the side lobe of the OTF and suppress the defocused background of the reconstructed image. As shown in FIG. 3, a in the figure is | OTF & ltcalness & gt after SIM is combined2As a result (approximated by the denominator of equation 4), b in the figure is the theoretical | OTF2As a result, c in the graph is the gray value corresponding to the straight lines in a and b, where the arrow indicates | OTF tintafter SIM combination2And theory | OTF-2With significantly different regions, OTF with m ═ 1 is modulated at the time of combining so that the | OTF after SIM combining is non-viable2(ii) Cure of theoretical | OTF2The distribution is similar, and the abnormal characteristics of the OTF after SIM combination can be modulated; and the OTF corresponding to the molecule m being 0 in step 4 is also subjected to filtering processing, and the filtering processing has the effect of removing the out-of-focus background of the image.
In one embodiment, step 4 is achieved by equation (4):
wherein S represents the SIM super-resolution image outputted in step 4, FilterdenominatorRepresenting a second Filter, FilternumertorDenotes a third filter, k ═ k (k)x，ky) Wherein k isxAnd kyRespectively represent the image frequency domain space coordinates, Om(k+mpd) Representing optical propagation functionO (k) according to mpdAs a result of the shift, the result is,
representing said filtered spectral components output by said step 2
According to mp via said step 3dAs a result of the shift, d is 1, 2, 3, m is 0, +1, -1, a (k) denotes an apodization function, w denotes reconstruction adjustment parameters, w denotes>0, for example w ═ 2, ifft {. cndot.) denotes the inverse fast fourier transform.
In one embodiment, step 4, FilterdenominatorIs set to the formula (5), FilternumertorIs set to formula (6):
wherein k is (k)x，ky) Wherein k isxAnd kyRespectively representing the spatial coordinates of the frequency domain of the image, a, b and c are all the adjusting parameters of the filter, 0<a≤1，b>0，c>0, normally: a is 0.5, b is 1, c is 1,
representing the square of the vector two norm.
In one embodiment, step 4, FilterdenominatorIs set to the formula (7), FilternumertorIs set to formula (8):
in one embodiment, step 4, FilterdenominatorIs set to the formula (9), FilternumertorIs set to formula (10):
the quality enhancement system for the structured illumination light super-resolution microscope image comprises a frequency spectrum separation unit, a first filter, a frequency spectrum moving unit and a reconstruction image unit, wherein:
the spectrum separation unit is used for carrying out spectrum separation on the original SIM image to obtain different spectrum components.
The first filter is used for carrying out frequency domain filtering on the frequency spectrum component and removing the defocusing information distributed in the low-frequency region of the frequency spectrum to remove the streak artifact and obtain the frequency spectrum component
Spectrum mobile unit for using said
According to mpdMoving to obtain the moved frequency spectrum component
A reconstruction image unit for reconstructing the image
Performing a spectral groupingCombining; meanwhile, the optical propagation function is modulated to inhibit the defocused background, and finally, an SIM super-resolution image is output.
In one embodiment, the first filter NF is set to equation (1) or (2) or (3):
in the formula (I), the compound is shown in the specification,
wherein k isxAnd kyRespectively representing the spatial coordinates of the frequency domain of the image, n representing the order of the filter, a, b and c being the adjusting parameters of the filter, 0<a≤1，b>0，c>0，n>0, normally: a is 0.5, b is 1, c is 1, and n is 6.
In one embodiment, the reconstructed image unit is arranged as equation (4):
wherein S represents a super-resolution reconstructed image output by the reconstructed image unit, FilterdenominatorRepresenting a second Filter, FilternumertorDenotes a third filter, k ═ k (k)x，ky) Wherein k isxAnd kyRespectively representing the spatial coordinates of the image frequency domain, Om(k+mpd) Representing the optical propagation function O (k) in terms of mpdAs a result of the shift, the result is,
representing the filtered spectral components of the first filter output
By mp via said spectrum mobile unitdAs a result of the shift, d is 1, 2, 3, m is 0, +1, -1, a (k) represents an apodization function, and w (w)>0) Indicating the reconstruction adjustment parameters. In actual calculation, w is usually 2, and ifft {. cndot. } represents the inverse fast fourier transform.
The Filterdenominator、FilternumertorTo equations (5) and (6), respectively, or to equations (7) and (8), respectively, or to equations (9) and (10), respectively:
finally, it should be pointed out that: the above examples are only intended to illustrate the technical solution of the present invention, and not to limit it. Those of ordinary skill in the art will understand that: modifications can be made to the technical solutions described in the foregoing embodiments, or some technical features may be equivalently replaced; such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (8)
1. A method for enhancing the quality of a structured illumination light super-resolution microscope image is characterized by comprising the following steps:
step 1, carrying out frequency spectrum separation on an original SIM image to obtain different frequency spectrum components;
step 2, carrying out frequency domain filtering on the frequency spectrum component, removing the defocusing information distributed in the low-frequency region of the frequency spectrum to remove the streak artifact and obtain the frequency spectrum component
Where d denotes a direction of the structured illumination light, d 1, 2, 3, m denotes whether the spectral component is to be moved, m 0 denotes that the spectral component is not to be moved, and m ± 1 denotes that the spectral component distribution is in accordance with ± pdIs moved, pdRepresenting a structured illumination light wave vector, k ═ kx，ky)，kxAnd kyRespectively representing image frequency domain space coordinates;
step 3, mixing the above
According to mpdMoving to obtain the moved frequency spectrum component
Step 4, for the above
Carrying out frequency spectrum combination; meanwhile, modulating an optical propagation function to inhibit an out-of-focus background and finally outputting an SIM super-resolution image;
said step 2 filters said spectral components with a first filter NF described by equation (1) or equation (2) or equation (3):
in the formula (I), the compound is shown in the specification,
a. b and c are both adjusting parameters of the filter, and n represents the order of the filter.
2. The method for enhancing the quality of the structured-illumination light super-resolution microscope image according to claim 1, wherein the step 4 is realized by the formula (4):
wherein S represents the SIM super-resolution image output in the step 4, FilterdenominatorRepresenting a second Filter, FilternumertorDenotes a third filter, Om(k+mpd) Represents the optical propagation function O (k) in terms of mpdThe shifted result, A (k) represents the apodization function, w represents the reconstruction tuning parameters, and ifft {. cndot.) represents the inverse fast Fourier transform.
3. The method for enhancing the quality of the structured-illumination light super-resolution microscope image according to claim 2, wherein in the step 4, the FilterdenominatorIs set to the formula (5), FilternumertorIs set to formula (6):
wherein a, b and c each represent a filter adjustment parameter,
representing the square of the vector two norm.
4. The method for enhancing the quality of the structured-illumination light super-resolution microscope image according to claim 2, wherein in the step 4, the FilterdenominatorIs set to the formula (7), FilternumertorIs set to formula (8):
in the formula, b represents the filter adjusting parameter, | ·| non-woven phosphor2Representing the two-norm of the vector.
5. The method for enhancing the quality of the structured-illumination light super-resolution microscope image according to claim 2, wherein in the step 4, the FilterdenominatorIs set to the formula (9), FilternumertorIs set to formula (10):
in the formula, b represents the filter adjusting parameter, | ·| non-woven phosphor2Represents the two-norm of the vector, | ·| non-woven phosphor2Representing the two-norm of the vector.
6. A system for quality enhancement of structured illumination light super-resolution microscope images, comprising:
a spectrum separation unit for obtaining different spectrum components by performing spectrum separation on the SIM original image;
a first filter for performing frequency domain filtering on the frequency spectrum component to remove the defocus information distributed in the low frequency region of the frequency spectrum to remove the streak artifact and obtain the frequency spectrum component
Where d denotes a direction of the structured illumination light, d 1, 2, 3, m denotes whether the spectral component is to be moved, m 0 denotes that the spectral component is not to be moved, and m ± 1 denotes that the spectral component distribution is in accordance with ± pdIs moved, pdRepresenting a structured illumination light wave vector, k ═ kx，ky)，kxAnd kyRespectively representing image frequency domain space coordinates;
a spectrum shifting unit for shifting the frequency spectrum
According to mpdMoving to obtain the moved frequency spectrum component
A reconstruction image unit for reconstructing the image
Performing spectrum combining(ii) a Meanwhile, modulating an optical propagation function to inhibit an out-of-focus background and finally outputting an SIM super-resolution image;
the first filter NF is set to equation (1) or (2) or (3):
in the formula (I), the compound is shown in the specification,
a. b and c are both adjusting parameters of the filter, and n represents the order of the filter.
7. The system for quality enhancement of structurally illuminated light super-resolution microscope images according to claim 6, wherein the reconstructed image unit is set to equation (4):
wherein S represents the SIM super-resolution image output by the reconstructed image unit, FilterdenominatorRepresenting a second Filter, FilternumertorDenotes a third filter, k ═ k (k)x，ky) Wherein k isxAnd kyRespectively representing the spatial coordinates of the image frequency domain, Om(k+mpd) Represents the optical propagation function O (k) in terms of mpdA shifted propagation function, A (k) represents an apodization function, w represents a reconstruction adjustment parameter, ift {. cndot. } represents a fast Fourier inversionAnd (4) changing.
8. The system of claim 7, wherein the Filter is configured to enhance the quality of the structured-illumination light super-resolution microscope imagedenominator、FilternumertorTo equations (5) and (6), respectively, or to equations (7) and (8), respectively, or to equations (9) and (10), respectively:
wherein a, b and c each represent a filter adjustment parameter,
represents the square of the vector two-norm, | ·| non-woven phosphor2Representing the two-norm of the vector.
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